Figure
3.
A
novel
object
placed
within
the
open
field
apparatus
for
the
novel
object
exploration
task.

•
Elevated
plus
maze
(Figure
4)
constructed
from
black
opaque
acrylic,
with
each
arm
measuring
30x5
cm
and
the
central
platform
5x5
cm.
One
set
of
arms,
opposing
one
another,
are
enclosed
completely
by
a
wall
of
transparent
acrylic,
15
cm
high,
while
the
other
set
is
open
with
a
ledge
of
0.5
cm
either
side
of
the
arms.
The
maze
is
elevated
40
cm
from
the
ground
on
a
transparent
acrylic
stand.

•
Light-dark
box:
custom-built,
white
acrylic
(44x21x21
cm).
The
box
is
partitioned
unequally
with
a
sheet
of
white
acrylic,
21x50
cm,
such
that
approximately
one-third
(15x21x21
cm)
of
the
total
area
is
under
low
lighting
(10-15
lux)
representing
the
'dark
chamber,'
and
the
remaining
two
thirds
brightly
lit
(300-330
lux)
with
white
light,
which
served
as
the
'light
chamber'
(Figure
5).
A
small
opening
within
the
partition,
5x7
cm,
allows
mice
to
move
freely
between
chambers.

Figure
5.
Dorso-ventral
view
of
the
light-dark
box
showing
brightly
lit
'light
chamber'
and
the
'dark
chamber'
where
the
mouse
is
hiding.

•
Puzzle
box
apparatus
(Figure
6):
custom-built,
white
acrylic
(73x28x27.5
cm).
The
box
is
unequally
partitioned
into
2
compartments
with
a
movable
black
sheet
of
acrylic,
28x27.5
cm,
which
designated
a
"start-box"
on
one
side,
58x28x27.5
cm,
and
a
smaller
"goal
box"
on
the
other,
14x28x27.5
cm.
Two
different
partitions
are
used
in
this
test:
the
first
(partition
1)
has
an
entrance
cut
into
the
base
of
it,
4x4
cm,
which
enables
visibility
of-and
access
to-the
goal
box
from
the
start-box
(Figure
9,
day
1
and
trial
1);
the
second
(partition
2)
is
merely
a
partition.
An
underpass,
15x4x2
cm,
is
cut
into
the
2.5
cm
thick-base
of
the
apparatus,
spanning
the
partition
and
allows
mice
to
move
between
compartments
when
it
is
in
place.

•
A
blue
circular
container
made
of
plastic,
diameter
70
cm
and
depth
28
cm,
is
used
for
the
Morris
water
maze
test
(Figure
7).
The
container
is
divided
equally
into
quadrants
and
the
edge
marked
to
designate
the
following
for
detailed
tracking
in
EthoVision:
right
(R),
left
(L),
target
(T),
and
opposite
(O).
Each
quadrant
is
used
to
determine
the
placement
of
the
mouse
into
the
maze
which
alternated
for
successive
trials
(see
Figure
9
for
detailed
illustration).
Tap
water
(21-22°C)
is
used
to
fill
the
container
up
to
a
depth
of
22
cm,
giving
a
water
surface
diameter
of
60
cm.
A
non-toxic
whitener
(80
g
of
milk
powder)
is
used
to
color
the
water.
Light
intensity
across
the
surface
of
the
water
is
set
to
100-150
lux.
A
transparent
plastic
square
platform,
6x6x21
cm,
is
placed
in
middle
of
quadrant
T
(1
cm
above
for
the
visible
trials
and
1
cm
below
the
water
during
the
acquisition
phase)
for
all
trials
except
reversal.
For
the
reversal
session,
the
platform
is
submerged
1
cm
in
the
middle
of
quadrant
O.

Figure
7.
Above
view
of
a
Morris
water
maze
container
with
a
visible
platform.

•
Tail
suspension
apparatus:
A
cord
(3
mm
diameter)
is
extended
and
secured
between
two
legs
of
an
upturned
chair,
at
least
30
cm
height
from
the
base
(Figure
8).

Cardboard
cones

Soft
padding

Figure
8.
Schematic
illustration
of
the
tail
suspension
test.

Reagents,
supplies,
solutions

70%
alcohol

Disinfectant
solution:
1%
Trigene®

Paper
towels
and
other
cleaning
materials

Procedure
for
conducting
a
battery
of
behavioral
tests

Procedures
common
to
all
behavioral
testsa.
The
battery
of
behavioral
tests
is
conducted
in
order
from
the
least
to
most
stressful
(see
workflow
above).
b.
Each
apparatus
is
wiped
clean
with
1%
Trigene®
between
subjects
to
avoid
olfactory
cueing
influencing
activity
and
behavioral
performances.c.
Mice
are
tested
in
a
pseudorandom
order
and
are
moved
to
the
behavioral
suite
adjacent
to
the
housing
room
immediately
before
testing
with
a
minimal
transfer
time.d.
Behaviors
for
all
tests
are
recorded
and
archived
on
videotapes
for
more
detailed
analysis.e.
A
comparable
method
of
hand
coding
is
used
to
score
locomotor
activity
in
the
OF
and
LD
tests
for
albino
mice,
since
automated
tracking
for
these
mice
is
not
readily
accessible
using
EthoVision
software.
f.
Mice
are
returned
to
their
home
cages
at
the
end
of
each
test.

II-Open
field
a.
During
testing,
white
light
of
300-330
lux
intensity
is
used
to
evenly
illuminate
the
entire
open
field
arena.b.
At
the
start
of
each
test
session
a
mouse
is
taken
from
its
home
cage
and
placed
into
a
corner
of
the
open
field
arena,
such
that
it
is
facing
the
wall.
c.
Open
field
activity
is
then
video-recorded
for
5
min
for
further
analysis
using
EthoVision.d.
At
the
end
of
each
open
field
test,
the
number
of
fecal
boli
and
urine
puddles
are
recorded.e.
In
order
to
determine
center
locomotor
activity
(cm),
duration
(s)
and
frequency
in
the
open
field,
horizontal
movement
within
a
square
of
equal
distance
from
the
periphery
or
within
the
designated
"central
zone"
(36x36
cm)
is
tracked
using
EthoVision
(see
Figure
2).f.
Latency
(s)
to
enter
the
center
as
well
as
peripheral
locomotor
activity
(cm)
are
also
measured
using
EthoVision.

III-Novel
object
exploration
a.
The
novel
object
test
is
performed
48
h
following
the
open
field
test
using
the
same
apparatus
(see
above
for
details)
to
measure
the
exploratory
activity
of
a
mouse
in
response
to
a
novel
object
placed
within
the
center
of
the
arena.b.
During
each
test
session
a
novel
object,
consisting
of
a
blue
metal
cylinder,
20
cm
height
and
7
cm
diameter
with
a
white
top
surface
for
tracking
(see
Figure
3),
is
introduced
into
the
arena.c.
During
testing,
low
white
lighting
of
approximately
30
lux
intensity
is
used
to
evenly
illuminate
the
entire
open
field
arena.d.
As
in
the
open
field
test
above,
a
mouse
is
taken
from
its
home
cage
and
placed
into
a
corner
of
the
open
field
arena,
such
that
it
is
facing
the
wall
at
the
start
of
each
test
session.e.
The
mouse
is
allowed
to
habituate
to
the
testing
condition
before
a
novel
object
is
placed
in
the
center
of
the
open
field
arena
2
min
into
the
test
session,
after
which
the
mouse
is
allowed
to
explore
the
object
freely
for
the
remaining
3
min
of
the
test.f.
A
circular
area
(measuring
20
cm
in
diameter)
around
the
object
is
defined
and
used
as
the
"exploration
zone"
in
EthoVision
recorded
tracking
(see
Figure
3).g.
During
the
final
3
min
of
exploratory
activity,
latency
to
initial
exploration
of
the
novel
object,
as
well
as
the
frequency
and
duration
of
exploration
around
the
object,
and
rearing
behaviors
are
recorded.

IV-Elevated
plus
mazea.
Light
intensity
around
the
maze
is
set
between
300
and
330
lux.b.
At
the
start
of
every
elevated
plus
maze
(EPM)
task,
a
mouse
is
placed
on
the
central
platform,
facing
towards
a
closed
arm,
and
allowed
to
explore
the
maze
freely
for
5
min.c.
At
the
end
of
each
elevated
plus
maze
test,
the
number
of
fecal
boli
and
urine
puddles
are
recorded.d.
To
score
behaviors
from
videotapes
of
the
EPM,
EthoLog
version
2.25
is
used.

-When
all
four
paws
enter
an
arm,
an
arm
entry
is
counted;
similarly,
when
the
forepaws
are
out
on
an
arm,
a
central
entry
is
counted.-An
arm
exit
is
considered
when
all
four
paws
leave
the
arm.

e.
The
following
behavioral
indices
are
scored:
open
and
closed
arm
entries;
open
and
closed
arm
duration;
latency
to
first
enter
an
open
arm;
and
time
spent
on
the
central
platform
after
initial
placement.

V-Light-dark
box
a.
Each
mouse
is
taken
from
its
home
cage
and
placed
into
the
dark
chamber
facing
the
end
wall
(parallel
to
the
partition)
except
for
the
group
that
is
used
to
investigate
initial
placement
in
the
light,
which
are
placed
facing
the
end
wall
(parallel
to
the
partition)
within
the
light
chamber.
b.
Activity
in
the
light-dark
box
is
video-recorded
for
5
min.c.
At
the
end
of
every
light-dark
box
test,
the
number
of
fecal
boli
and
urine
puddles
are
recorded.d.
The
latency
for
each
mouse
to
emerge
from
the
dark
chamber
into
the
light
chamber
is
recorded;
for
the
group
placed
in
the
light
chamber
at
the
start
of
the
test,
latency
to
move
into
the
dark
is
likewise
recorded
using
EthoLog.e.
Duration
in
each
chamber
and
the
number
of
light-dark
transitions
are
also
hand
coded
using
Etholog.
A
single
transition
is
counted
when
all
four
paws
had
enter
a
chamber.f.
Activity
within
the
dark
chamber
and
light
chamber
are
measured
using
Ethovision,
and
mean
speed
is
derived
from
these
measures.

VI-Puzzle
box
The
puzzle
box
is
used
to
demonstrate
the
motivation
of
a
mouse
to
solve
problems
when
exposed
to
a
brightly
lit
arena.
It
is
adapted
from
the
problem
solving
paradigm
for
rats
in
which
a
series
of
tasks
are
presented
in
order
of
difficulty.
Previous
results
from
submitting
investigator's
laboratory
suggested
that
"mice
employ
contextual
memory
and
spatial
navigation
to
solve
problems
within
the
puzzle
box."a.
During
testing
light
intensity
is
set
between
600
and
700
lux
across
the
start-box,
while
the
goal
box
is
covered
with
a
white
acrylic
sheet
to
minimize
illumination
at
<1
lux.b.
A
mouse
is
placed
within
the
start
box
and
the
latency
to
move
into
the
goal
box
via
the
underpass
is
recorded
over
successive
trials.
c.
The
puzzle
box
test
is
accomplished
in
3
consecutive
days
starting
with
a
training
session
on
day
1
(see
Figure
9
below):

Training
Session
Day1/trial
1:
only
partition
1
is
in
place
(see
details
above)
to
separate
the
start-box
from
the
goal
box,
through
which
the
goal
box
is
visible.Day1/trials
2
and
3:
the
partition
2
is
in
placed
for
the
remaining
trials
(see
Figure
9),
the
purpose
is
for
the
mouse
to
learn
how
to
access
the
goal
box
via
the
underpass.
Burrowing
SessionDay2/trial
1:
the
mouse
is
prompted
to
enter
the
goal
box
via
the
open
underpass.Day2/trials
2
and
3:
the
underpass
is
then
filled
with
sawdust,
such
that
the
mouse
is
prompted
to
burrow
in
order
to
reach
the
goal
box.

Plug
SessionDay3/trial
1:
the
mouse
is
prompted
to
enter
the
goal
box
via
the
open
underpass.
Day3/trials
2
and
3:
the
underpass
is
"plugged"
across
with
a
rectangular
piece
of
corrugated
cardboard
(7.5x2.5x0.5
cm)
mounted
in
place
by
a
cardboard
block
(2x3x1.5
cm),
and
the
mouse
is
prompted
to
learn
to
remove
the
plug
in
order
to
access
the
goal
box.

d.
After
initial
training
session
on
day
1,
the
puzzle
test
is
continued
with
two
different
problem
solving
sessions
on
days
2
and
3
(Figure
9).
e.
For
each
test
session,
3
trials
with
increasing
level
of
difficulty
are
carried
out
in
succession.f.
Each
mouse
is
taken
from
its
home
cage
and
placed
into
the
start-box
facing
the
end
wall
parallel
to
the
partition.g.
The
latency
to
enter
into
the
goal
box,
or
when
all
four
paws
are
in
place,
is
recorded
for
each
trial.h.
The
mouse
is
returned
to
its
home
cage
between
trials,
or
after
a
maximum
of
3
min
trial
duration.
i.
Mean
latencies
are
calculated
for
the
training,
burrowing
and
plug
sessions
as
an
index
of
the
problem
solving
abilities
of
each
mouse
across
the
given
tasks.

Figure
9.
Scheduled
tasks
for
the
puzzle
box.
Day
1
initial
training
phase
for
learning
acquisition,
followed
by
Day
2
problem
solving
burrowing
task,
and
Day
3
problem
solving
plug
task.
Underpass
is
"open"
when
it
is
not
obstructed.
Underpass
is
"closed"
when
it
is
blocked
or
obstructed
by
sawdust
or
solid
plug.

VII-Morris
water
maze
a.
Each
mouse
is
trained
to
remember
the
location
of
the
initially
visible
platform
that
is
subsequently
submerged
in
water
during
acquisition.
A
reduction
in
the
latency
to
localize
the
platform
over
successive
trials
is
indexed
as
learning.b.
Each
mouse
is
taken
from
its
home
cage
and
placed
into
the
water
maze
at
the
designated
quadrant
facing
the
wall,
and
allowed
a
maximum
of
60
s
to
locate
the
platform.
A
quadrant
sequence
is
pre-determined
for
placement
of
the
mouse
into
the
maze
across
trials
per
session
(see
Figure
10
for
detailed
illustration).c.
After
60
s
in
the
water
maze,
the
mouse
is
guided
to
the
platform
with
the
use
of
a
wooden
plank
(26x4.5x1
cm),
and
returned
to
its
home
cage.d.
The
inter-trial
interval
is
set
at
approximately
6
min.e.
On
the
final
test
day
of
reversal
session
or
"reversal"
learning
task,
the
platform
is
submerged
1
cm
in
the
middle
of
quadrant
O
(opposite),
whereby
the
location
of
the
platform
is
moved
to
a
different
quadrant
(O)
of
the
pool,
thereby
measuring
the
flexibility
of
spatial
orientation
via
the
ability
to
rapidly
and
accurately
learn
the
new
location
of
the
platform.
f.
Latency
to
reach
the
platform
with
all
four
paws
is
recorded
for
each
mouse:

•
Training
Phase
Day1/Session
1:
the
platform
is
visible
1
cm
above
the
surface
of
the
water,
for
each
trial.

•
Reversal
TaskDay1
to
Day3/all
trials
in
Sessions
2-6:
the
platform
is
submerged
1
cm
beneath
the
surface
of
the
water.Day4/Sessions
7
and
8:
the
platform
is
placed
in
quadrant
O,
diagonally
opposite
the
original
placement
quadrant
T
(see
Figure
10
below);
and
the
wooden
plank
is
initially
placed
on
the
platform
for
trial
1
of
session
7
to
indicate
its
new
location.

g.
In
order
to
obtain
session
performance
per
mouse,
mean
latencies
are
calculated
across
the
trials
per
session
for
each
mouse.
The
Morris
water
maze
testing
is
accomplished
within
4
consecutive
days,
with
2
sessions
(morning,
afternoon)
per
day,
each
session
consisting
of
4
trials
for
a
final
total
of
32
trials.

Figure
10.
Morris
water
maze
test
schedule.
The
maze
is
divided
into
4
equal
quadrants
as
indicated
by
each
circle:
right
(R),
left
(L),
target
(T),
and
opposite
(O)-
used
to
designate
the
initial
placement
of
the
mouse
into
the
maze.
A
sequence
is
pre-determined
and
remained
the
same
across
morning
and
afternoon
sessions.
A
square
platform
is
placed
in
the
T
quadrant
for
all
trials
on
days
1-3,
and
is
placed
in
quadrant
O
for
the
reversal
sessions
on
day
4.
The
visible
(black
square)
platform
is
1
cm
above
the
surface
of
the
water
during
training
in
session
1,
and
1
cm
below
the
surface
during
acquisition
(gray
square)
in
sessions
2-6.
Schematic
illustration
is
not
drawn
to
scale.

VIII-Tail
suspension
testa.
Two
separate
trials
of
the
tail
suspension
test
are
performed
on
each
mouse
at
approximately
the
same
time
of
day
on
consecutive
days,
with
a
minimum
of
24
h
between
trials.
b.
A
cardboard
cone
is
placed
around
the
tail
of
each
mouse
immediately
before
the
test,
with
its
tail
extending
through
the
tip,
to
prevent
tail
climbing
behaviors.
c.
The
mouse
is
suspended
at
approximately
one-third
from
the
end
of
its
tail,
using
soft
padding
around
the
area
to
protect
the
tail,
and
plastic
clothes
pegs
are
used
to
secure
the
mouse
to
the
line
(Figure
8).d.
Each
trial
is
5
min
long
and
analyzed
using
the
mobility
detection
module
in
EthoVision.

Definitions

The
novel
object
test
is
performed
using
the
same
apparatus
as
the
open
field
test
to
measure
exploratory
behavior
in
the
absence
and
presence
of
a
novel
object
placed
within
the
center
of
the
arena.

Puzzle
box
is
adapted
from
the
problem
solving
paradigm
for
rats
(Lad
et.
al.,
2010)
in
which
a
series
of
tasks
are
presented
in
order
of
difficulty.
The
puzzle
box
is
also
devised
to
demonstrate
the
motivation
of
a
mouse
to
solve
problems,
employing
contextual
memory
and
spatial
navigation,
when
exposed
to
a
brightly
illuminated
arena
for
which
it
is
driven
to
escape.

The
Morris
water
maze
test
is
designed
to
test
the
spatial
learning
ability
and
memory
of
a
mouse
across
several
trials,
run
over
a
number
of
days,
using
visual
cues
placed
around
a
water-filled
maze.
Initially
a
mouse
is
trained
to
remember
the
location
of
a
visible
platform
that
is
subsequently
submerged
in
water
during
acquisition
phase.
Learning
index
is
deduced
from
reduced
latency
to
locate
platform
over
successive
trials.
On
the
final
day
a
"reversal"
learning
task
is
included
to
measure
the
flexibility
of
spatial
orientation
via
the
ability
to
rapidly
and
accurately
learn
the
new
location
of
the
platform.

Data
collected
and
submitted
by
investigator

Open
field
test:
total
activity
(center,
periphery,
rearing
in
presence
of
novel
object),
exploratory
behavior
in
the
absence
and
presence
of
a
novel
object
(center
square
occupancy).